JP4840593B2 - Optical resonance analysis unit - Google Patents

Description

  This application is relative to US Provisional Application Nos. 60 / 492,061 and 60 / 49,2062, both filed on Aug. 1, 2003, the disclosures of which are incorporated herein by reference. Claim priority.

  The present invention generally relates to optical resonance analysis units. In particular, the present invention provides an improved instrument for conducting grid-coupled surface plasmon resonance imaging utilizing an illumination system and a detection system for real-time analysis of multiple reactions traveling on the surface of a sensor array. About.

  The basic principle of operation of lattice-coupled surface plasmon resonance (GCSPR) utilizes surface charge oscillations that are generated when light of a specific wavelength strikes a metal surface. For example, a sensor chip that typically includes a plastic optical grating coated with a thin (up to 80 nm) layer of a highly reflective metal such as gold is interspersed with an array of specific binding molecules (eg, antibodies). ing. When the chip is illuminated with light of the appropriate wavelength, polarization, and angle of incidence, a resonant state is created as energy from the light couples into the metal electrons to excite surface plasmons. Therefore, this resonance state is a propagation oscillation of free electrons in the metal at the metal / dielectric interface. In this example, the metal is a gold layer and the dielectric is an aqueous solution containing molecules to be analyzed that are flowed across the metal surface in contact with the immobilized binding molecules. Surface plasmons have an electric field perpendicular to the interface and propagate along this electric field. The amplitude of the plasmon electric field is maximized at the interface and decays exponentially perpendicular to the interface. The transmittance to the dielectric depends on the wavelength of the excitation light and is typically in the range of 100 to 300 nm. The resonance state is clarified by a large drop in the reflectance of the incident beam (when incident light is coupled into the surface plasmon).

  As molecules from the solution bind to the material deposited on the metal surface, the refractive index of the deposited material changes, which in turn causes a shift in the SPR resonance angle. SPR can be used for detection of molecular binding reactions on the surface of the sensor chip. This is because the SPR resonance state depends on the refractive index at the metal / dielectric interface. Thus, the molecular binding reaction on the surface of the sensor chip causes a shift in resonance, which can be monitored as a shift in wavelength, angle of incidence, or intensity, depending on the implementation.

  However, current grid-coupled SPR methods that employ angular scanning array imaging to measure many samples in parallel are not compatible with other angular scanning optical resonance sensor methods, including Kretchmann's SPR imaging method. Share the disadvantages. Kretschmann, Z.M. See Physik, 241: 313-24 (1971). Many of the problems with angular scanning arrays SPR require optics and an imaging system with a relatively large numerical aperture to accommodate the range of illumination angles that SPR array imaging involved in SPR scanning, Associated with the fact that for each individual exposure or image frame during a single angular scan, the light is highly concentrated in a small portion of the full aperture pupil.

  The conventional lens design used for SPR detection analysis exposes a scan angle dependent image defect known as “walking” or “ROI shift” in which the area of interest on the sensor chip is analyzed. The (ROI) image moves across the detector surface as the illumination angle (incident angle) is scanned. Walking is particularly evident in the area surrounding the field. This effect is due to the combination of higher order aberrations that depend on both the aperture angle and the field radius, and is facilitated when the object plane is tilted. Although such aberrations are common in conventional high numerical aperture imaging systems, they are generally acceptable and only cause a loss of contrast or resolution when all aperture angles are present simultaneously. is there. However, in an array SPR, such aberration presents a serious problem. This is because highly accurate reflectance measurements must be made at carefully defined locations on the sensor chip surface depending on the illumination angle. Only a small part of the lens aperture pupil is used for any single exposure, but the adopted part moves across the aperture during angular scanning, thereby changing the image position on the detector To do.

  The ROI shift effect can be compensated to some extent in software by moving the detector pixels containing the ROI as a function of scan angle to track the movement of the ROI image. Zizlsperger and Knoll, Progr. Colloid Polym. Sci. 109: 244-253 (1998). However, such a posteriori data processing compensation for the walking effect is, first of all, a less favorable solution for the generation of more accurate data.

  Another problem with the aforementioned severe momentary underfilling of aperture pupils in current SPR array instruments is the “hot spot” or image caused by multiple reflections between various optical surfaces, particularly between lens element surfaces. It is a flare phenomenon. Such multiple reflections cause stray light and some loss of contrast in all multi-element refractive imaging systems, even with the use of anti-reflective coatings, but this effect is generally benign. is there. However, in an angular scanning SPR imaging system, a high concentration of light intensity in a small portion of the aperture pupil results in stray light that is concentrated in a relatively small portion of the image field on the detector surface. These concentrated compartments are “hot spots”. In addition, each hot spot generally moves across the image as the illumination angle is scanned. Although the intensity of each hot spot is a very small portion of the direct intensity on the detector, the hot spot can significantly modulate the apparent drop in reflectivity associated with the affected ROI. An affected ROI has a widely varying SPR resonance angle compared to an unaffected ROI, which therefore results in increased background noise in the system.

  Other methods in the literature use imaging, but imaging suffers from scanning-dependent spurious signals or otherwise scanning is totally avoided. For example, Zizlsperger and Knoll, infra. Describes a system in which angular scanning results in enormous walking that has been carefully handled via ROI software tracking. Guedon et al. Describe a system that compensates for poor imaging and also a huge walk, but compensates through the use of a fixed angle and a relatively small and large ROI. Guedon et al., Anal. Chem. 72: 6003-6009 (2000) and Lyon et al., Review of Scientific Instruments, 70 (4): 2076-2081 (1999). Another approach is the double grating normal incidence imaging of Knoll's patent US Pat. No. 5,442,448, but this system introduces additional complexity. However, fixed angle and wavelength systems have a very limited dynamic range and are susceptible to light source intensity variations. For example, none of the above-described techniques can obtain a sufficiently good image to read a logo or other small identifier or indicator structure, or even a small ROI on the sensor surface, even using software compensation . As contemplated herein requiring detection of resonance angles for an array of target ROIs and reference ROIs with a comparison of target ROIs indicating immobilized reactants relative to a reference ROI indicating a bare metal sensor surface A large dynamic range is required in the system. That is, it is necessary to be able to detect a resonance angle that varies widely. Such an application can be used to compensate for system variations that affect both types of ROI, such as changes in temperature, pressure, and internal index of refraction, of the target ROI and reference ROI resonance angles. Requires deduction.

Steiner et al., In Journal of Molecular Structure, 509: 265-273 (1999), are looking at improving SPR image quality by tilting the detector CCD chip. While achieving the objective of correcting for the simple defocus found for static non-vertical incidence angle SPR imaging systems, the improvement is the wide range required for array sensors with significant dynamic range. It does not fully address the “walk” problem of images when collecting images over the angle of incidence. Although the Steiner et al. Instrument did not continuously angle scan, it collected images only at a single fixed angle, so the “residual walk” described in this application remained unnoticed.
U.S. Provisional Application Nos. 60 / 492,061 and 60 / 49,2062 both filed on August 1, 2003 US Pat. No. 5,442,448 to Knoll Kretschmann, Z.M. Physik, 241: 313-24 (1971) Zizlsperger and Knoll, Progr. Colloid Polym. Sci. 109: 244-253 (1998). Zizlsperger and Knoll, infra. Guedon et al., Anal. Chem. 72: 6003-6009 (2000) Lyon et al., Review of Scientific Instruments, 70 (4): 2076-2081 (1999). Steiner et al., Journal of Molecular Structure, 509: 265-273 (1999).

  The present invention is the first invention that describes and corrects for this unexpected residual walk effect that was not previously known in the art.

  Surprisingly, the present invention solves these optical problems through a complete set of “angular scan compensated imaging” techniques, which include the proper tilting of the detector (CCD) chip, Includes special optimization of imaging optics, corrector plates, and special alignment techniques to minimize ROI shift.

  Thus, the present invention is an improved optical resonance suitable for performing lattice coupled SPR analysis and useful for simultaneously monitoring an array having hundreds of isolated reaction regions (ie, target ROIs) on the sensor surface. Intended for analytical instruments. As explained below, the present invention relies on the Kretschmann-type SPR analysis described above, and has a limited number of available reaction loci, as well as measurement accuracy and dynamic range. Provides several advantages over current equipment.

  In addition, the instrument is designed to solve many of the aforementioned problems that currently exist in the field of lattice coupled SPR array analysis, and therefore represents a major advance in the field.

  Accordingly, the present invention is directed to an improved optical resonance analysis imaging apparatus for use in grating coupled surface plasmon resonance (GCSPR). In particular, the present invention provides system fluid dynamics, temperature control, sensor scanning, as well as from scanned sensors, in addition to providing real-time simultaneous analysis of up to thousands of molecular binding interactions on the sensor surface. It combines many features that also provide improved control of reaction parameters involved in data collection and analysis. In addition, the present invention is designed to optimize angular scan range, angular accuracy, image fidelity, and eliminate resonant spurious signals. In addition, the present invention also describes a novel method for monitoring reactions that occur on the surface of a sensor utilizing the novel instrument described herein.

  Included with the novel features of the present invention is a significant improvement in the image over the entire field while minimizing image movement (“walk” or “ROI shift”) as the beam angle is scanned. This is a novel relay lens design that, in turn, greatly improves the overall resolution of the scanned sensor surface. In order to achieve this surprising image improvement, the instrument described herein is capable of precise alignment of the entire integrated optical system, in particular the sensors, light sources, and It is important that alignment between detectors (such as a CCD camera) can usually be achieved.

  More particularly, according to the present invention, important aspects of the interaction between system components are the focal position of the camera lens, the distance between the detector and the sensor surface, and the detector relative to the sensor surface. Including the tilt angle. The mechanical functions of the instruments described herein are interconnected in such a way that the necessary adjustments required to optimize the performance of the optical unit are performed in concert. These mechanical adjustments analyze important data and quickly adjust various parameters to quickly optimize instrument calibration to improve image quality and reduce optical image aberrations Are preferably executed with the assistance of image analysis software specially designed to assist.

  In one aspect, the present invention is directed to a fully housed and integrated optical grating coupled surface plasmon resonance analysis unit. The unit includes a support frame with a target area designed to receive the sensor. In one embodiment, the sensor can be a lattice-coupled surface plasmon resonance chip suitable as a substrate on which thousands of molecularly coupled reaction loci can be precisely arranged for simultaneous optical analysis. The unit can be designed so that the sensor can be inserted into the target area either mechanically or manually.

  The present invention includes a light source for directing illumination onto the sensor, which is then reflected from the sensor surface to the novel detector assembly described in more detail below. In each embodiment of the present invention, the sensor remains stationary and the light source allows the beam of light that goes out of the light source and directed onto the reflective sensor to move through multiple angles with respect to the sensor. Is mounted on the equipment frame. For example, in a preferred embodiment, the light source is pivotally secured to the frame such that a beam of light exiting the light source strikes the sensor at multiple angles. In a particularly preferred embodiment, the light source is mounted on the frame of a unit housed in such a way that the light source is movable in an arcuate path, so that the light source is many in a bi-directional pattern, ie positive and negative directions. The target area can be illuminated from different angles. The sensor surface is positioned at the optical apex of the swirling light source to minimize intensity fluctuations moving across the sensor that can lead to increased signal noise.

  In one embodiment, the light source includes a light emitting diode (LED) assembly for generating illumination that is directed to the sensor. By way of example only, the LED assembly can include a single 875 nm LED mounted on a printed circuit board and can include an independent aperture element to block unwanted light, but is widely suitable There are many variations on this design, including the LED wavelength. The LED lens assembly of the present invention is designed to mimic a point light source as much as possible, thereby minimizing any stray light that leaves the LED die itself. This stray light can lead to a less collimated light source beam and can cause a large number of light source points, both resulting in an increase in resonance width and subsequently an increase in SPR angular noise. The front surface of the LED assembly is dome-processed by rubbing this front surface into an optically flat surface to direct all rays through the aperture in a predictable path and into the collimating lens. The shape can be advantageously modified.

  In addition, the light source of the present invention also includes a light source optics assembly for controlling and optimizing the characteristics of light impinging on the sensor from the light source. In one embodiment, the light source optics assembly is housed in a lens tube and is suitable for collimating illumination exiting the light source, an interference filter for blocking unwanted wavelengths, and P Further comprising means for generating polarized light. In one embodiment, the filter width is 4 nm, which is selected to prevent excessive broadening of the SPR resonance profile and also prevents coherence noise (“speckle”). In another embodiment, a second polarizing filter can be added to generate S-polarized light (or alternatively, the current polarizer can be rotated 90 degrees), Thereby, the influence of the change in intensity can be canceled. The lens tube also provides a means for focusing the collimating lens.

  In another embodiment, the light source can be pivotably mounted directly to the instrument frame, or alternatively, can be brought into contact with the frame via a pivot arm. The swivel arm can include a support frame that is pivotally mounted to the frame of the unit. In this embodiment, the swivel arm includes means for fixing the light source, and means for connecting the light source to a swivel arm such as a motor and drive means for moving the light source.

  In a preferred embodiment, the light source is in contact with and repositioned by a stepper motor that can be mounted directly to the light source or fixed to the frame of the unit. In one embodiment, the sensor surface is attached to the light source at one end, and the action of the motor drives the arm back and forth, followed by the center of the sensor surface about the axis parallel to the groove of the sensor grid. The motor is fixed to the frame of the unit, and via the linear arm to allow the linear arm fitted with the motor to pivot substantially through In contact with the light source. In one embodiment, the arm is threaded and is moved by the rotational action of the motor.

  In an alternative embodiment, the instrument described herein can include a linearly scanned LED, the lens system of the light source can be rigidly secured to the frame, and the LED is driven by a motor Can be mounted on a movable linear slide. The movement of the LED changes the angle at which the collimated light strikes the sensor. This embodiment has a smaller motor, required to reposition the source beam through the desired range of angles, a shorter distance, less required for the LEDs to traverse to provide the same dynamic range. It has several advantages, including reduced vibration from mass and lower cost for equivalent scanning accuracy.

  In another embodiment utilizing a rotating mirror, the light source optics and LEDs can be fixed on a frame and can shine light on a rotating mirror. In this particular embodiment, the instrument includes means for precisely controlling the angle of incidence of the mirror. The advantages of this particular embodiment are as described above for linear LEDs.

  In yet another embodiment, a linear array of light source optics can be mounted on the frame and pointed towards a target area that includes a sensor. The geometric positioning of the source optics, i.e. the distance from the sensor and the distance between them, establishes and adapts to a sufficient number of intensity data points to establish and fit the SPR curve. Create the minimum required angle of incident light between any two adjacent optical systems. The light source optics can be illuminated sequentially very quickly to increase the data collection speed. An important advantage of this system is that it is in a solid state, has a stable geometry, and results in increased data rates.

  An additional variant is to keep all the light source optics illuminated and to pass through the movable aperture over the optics at a precisely controlled rate so that which rays strike the sensor. As well as limiting the exposure time by the aperture.

In one embodiment of the present invention, the collimated light generated by the light source strikes the sensor surface at a range of angles and is positioned to receive reflected light from the sensor from this surface. Reflected to the detector assembly. In one embodiment, the detector is mounted on the frame and is oriented to receive light reflected from the sensor surface. The reflected light changes in intensity as the reaction proceeds at each ROI on the surface of the sensor as the sensor scans through the range of angles. The signal output for each image frame is a measurement of two values for each ROI, (1) the intensity of the reflected light received by the detector and summed across each detector pixel including the ROI, and (2) reflected The intensity is converted to a corresponding incident angle measured as recorded by the angle encoder. As each angular scan is completed, the collected information is then transferred to a resonance qualification algorithm, which is preferably an Empirical Profile Fit (EPF) algorithm. In the fitting algorithm, the data described above for each ROI is fitted to a calibrated SPR profile, and the position of the minimum value of the curve is determined for each ROI. Subsequently, these resonance positions are tracked according to time by means of continuous angle scanning over the course of the movement execution. Real-time monitoring of SPR minimums with the novel instrument described herein provides important information regarding the reactions that are proceeding on the surface of the sensor in each of up to thousands of individual ROIs. In particular, for biomolecular sensing, the time dependence of the SPR response is dependent on the kinetic constants k _on and k _off that describe each ongoing binding and dissociation interaction in each ROI on the sensor surface. Enable calculation.

  In a preferred embodiment, the novel detector described herein includes a novel lens assembly for receiving light reflected from the sensor surface, a charge coupled device (CCD) camera, and a number of A multi-component gimbal seat assembly for adjusting the camera assembly in a plane is included. The novel detector assembly described herein preferably also includes a correction plate in the lens assembly to help reduce image aberrations contributing to ROI image shift (the “walking” effect), and In addition, a passive cold finger can be included to prevent deposition on the detector optics that can cause intensity variations.

  In a particularly preferred embodiment, the lens assembly is specifically designed for use in the novel optical resonance analysis unit described herein. Specifically, the lens assembly is specifically designed to maintain image stability on the CCD camera while the light source is scanned over a range of illumination angles. Sensor analysis using a conventional lens system is such that the target point moves or shifts with respect to the actual location of the target point (target ROI) on the sensor surface while “walking” or angular scanning proceeds. Bring the appearance. This leads to at least partial measurement of the reference ROI (bare metal) rather than only the target ROI, which in turn leads directly to an increase in measurement noise. The lens assembly of the present invention is designed to reduce this ROI shift effect, and the resulting SPR noise, thereby allowing more accurate reading and analysis of the reaction that is proceeding on the sensor surface. To do. The optical system of the present invention is further configured to provide accurate imaging across the sensor surface under conditions of off-axis motion or tilting of the object plane.

  The novel lens system described herein provides a magnification that is advantageously selected for projecting an image of the entire SPR sensor area onto a receiver optical sensitive area of a detector (eg, a CCD chip area). Including a sensor imaging lens having a monochromatic dual telecentric design. The optical system optionally includes a tilt corrector designed to be used with an appropriately tilted object (sensor) plane and (image (CCD detector) plane), the optical design comprising a sensor window, an aqueous The sample layer and detector camera window are fully considered, unlike conventional lens designs, the novel lens assembly of the present invention has an illumination scan angle (necessary for the large dynamic range described above). Designed specifically to minimize the image walk at the operating wavelength over the entire range, the lens assembly includes an objective section that produces an image of the tilted object plane at infinity, captures this virtual image, and This is followed by an image forming section that creates a reduced real image on the same tilted image plane that coincides with the two-dimensional detector surface, the optical section and the image forming section being located in the gap between the sections. Sharing intermediate opening plane passing. Novel aspects of the stop slot-shaped opening between the objective section and an image forming section of the plane is sandwiched. Lens assembly is described in further detail below.

  The optical analysis unit of the present invention preferably also includes a rotary encoder for precisely determining the incident angle of the light source described above, ie the angle at which the illumination strikes the surface of the sensor. The rotary encoder is preferably fixed in the unit and in contact with the light source in such a way as to determine the angle of the light source with respect to the sensor surface. This information is then transferred to a resonance qualification algorithm (e.g., EPF algorithm), where the data is fitted to calibrated SPR curves for various target points (ROIs), and the position of the curve minimum is determined. Tracked according to time. In a preferred embodiment, the rotary encoder includes a pivot shaft, an encoder housing, and an encoder.

  A linear encoder can be used as an alternative to the rotary encoder described above, and the linear encoder is used to track the position of the movable source optics as described above. Requires conversion from linear units to angular units. In another alternative embodiment, instead of a rotary encoder, a stepped motor mechanism that can directly determine the change in angle with a high degree of accuracy can be used.

  Each additional embodiment of the optical analysis unit of the present invention also includes a complex hydrodynamic system for transporting various fluid reagent solutions and sample solutions to and from the sensor surface. In one embodiment of the present invention, prior to performing an experimental operation, the unit's hydrodynamic system can be prepared by cleaning the system, flushing the system, and clearing bubbles from the system. This bubble can adversely affect the ability of the instrument to accurately collect and interpret the reaction proceeding on the sensor surface as well as the reflected optical data. Following contact with the sensor, the fluid system is designed to allow the user to direct the fluid to a waste receptacle, or alternatively, the fluid is re-contacted with the sensor or from the unit. It can be redirected through the system for either removal.

  In one preferred embodiment of the present invention, the hydrodynamic system has the ability to prepare both fluid paths simultaneously, as well as one specific fluid circuit while the SPR baseline (eg, for a buffer solution) is established simultaneously. Independent sample path and buffer path to allow the user the ability to prepare. Thus, the sample can be introduced into the system immediately prior to contact with the sensor surface, thereby rapidly degrading when it is removed from a sensitive sample, particularly optimal storage conditions such as low temperature anoxic storage. Maintain the integrity of samples that tend to

  In addition, in the preferred hydrodynamic system of the present invention, both the sample path and the buffer path are connected to an adjustable four-way valve that is positioned very close to the sensor. This valve functions to minimize sample / buffer mixing, which is important for accurate determination of the kinetic value.

  Additional features of the preferred hydrodynamic system include a sample injection station (SIS) that serves as a connection for sample entering the system and waste leaving the system. According to this feature, the sample valve is mechanically lowered into the solution, and then the sample is sucked up by the pump and injected into the sensor. The valve body can be operated mechanically or manually. SIS gives the user the option of discharging the sample for disposal, collecting the sample, or recirculating the sample across the sensor area. The ability to recirculate samples as described herein is particularly advantageous when a user can obtain only a small amount of a particular sample. The flexibility of the SIS described herein allows the user to connect to an automatic sampler (eg, a sample bank or carousel) for automatic injection of more diverse samples into the system. Can make it possible.

Another advantage of the most preferred hydrodynamic system described herein is the ability for “on-the-fly” sample loading, which includes two separate ones moving the buffer solution and one moving the sample. This allows the user to prepare the sample while the buffer is flowing through the system. In many cases, it is advantageous to use the instrument to allow the buffer solution to flow separately through the system for several minutes or more to establish a baseline. In addition, if sample loading is time dependent, for example for sensitive samples that may degrade over time and / or with temperature changes, the time between sample preparation, mixing, etc. and loading Independent buffers and sample hydrodynamics that samples are prepared and loaded just minutes or even seconds before injection and introduction onto the sensor Path is possible. Sample recirculation as described above is also advantageous, for example, for samples with particularly low on-rates (ie, low dissociation constants). Because this recirculation allows the sample to continue to (re) circulate through the system for a time long enough to observe a sufficient response to calculate the exact dissociation constant k _on Because it does.

  In addition, if the reaction proceeds slowly, recirculation is particularly advantageous in that a small amount of sample can be continuously recontacted with the sensor surface, whereas if this option is not available, the user Must obtain much larger samples, which can be difficult or possibly expensive if not impossible. Thus, this “infinite supply” of recirculated sample provides sufficient flexibility in both contact time and flow rate to allow monitoring of reactions that are very slow or limited in bulk delivery.

  The use of a four-way valve is particularly advantageous in the hydrodynamic system of the present invention. Because (1) the system flows simultaneously and prepares for sample injection, the four-way valve maintains the integrity of the buffer and sample concentration, and (2) the position of the four-way valve, ie immediately adjacent to the sensor This is because the position where it is kept minimizes any mixing at the buffer / sample interface.

  The hydrodynamic system of the present invention also includes a “bubble burst” fast pulsed flow driven by a syringe pump. When a new flow cell (including a new sensor chip) is operated, it is common for bubbles to remain in the cell gap after filling with a buffer. Bubbles may be introduced accidentally during sample switching and other fluid transfer operations. These bubbles interfere with the accurate measurement of the binding reaction on the affected ROI and must be removed. This removal can be difficult especially when the dimensions of the flow cell containing the sensor are reduced, as is common in the art of the present invention. The application of very high fluid flow rates beyond the critical evacuation flow rate (CDF) for short periods of time can remove trapped air bubbles due to non-uniform wetting tension on the flow cell surface or geometric discontinuities. Has been demonstrated.

  In the present invention, it has been determined that multiple applications of large flow pulses are usually necessary to eliminate bubbles while they move from one “sticky” location to the next “sticky” location. ing. To remove any bubbles from the flow cell, the hydrodynamic system is used to apply a “bubble rupture” or a series of high flow pulses to the sensor cell, for example via a small flow impedance fluid flow path It incorporates a positive displacement pump (syringe pump). In addition, real-time monitoring for bubbles can interrupt the hydrodynamic operation and “burst” any bubbles that may accumulate.

  In order to maintain the stability of the system and to generate accurate and consistent data regarding the binding reactions proceeding on the surface of the sensor, according to the present invention, the ambient temperature surrounding the sensor, as well as the sensor As such, it is particularly advantageous for the user to have some control over the temperature of the sample and solution in contact with the sensor, particularly to control the sensor surface on which the binding reaction under analysis proceeds. In addition, it is desirable to allow the user to perform experiments at various temperatures both above and below the ambient temperature. In order to facilitate the temperature control described above, the unit of the present invention may advantageously include a warming room that houses the target area where the sensor is located and that houses at least a portion of the hydrodynamic system described above. . In a preferred embodiment, the warming room includes a proportional integral derivative (PID) controlled thermoelectric module including a circulation fan, a heat sink, a thermal fuse, and a sensor. The warming chamber is preferably lined with an insulating foam that maintains the thermal stability within the warming chamber. This insulation protects the sensor area, chemical reaction, and incoming fluid against temperature fluctuations that can be caused by the environment or the operation of the equipment. Also preferably included in the warmer chamber is a passive preheater to maintain the thermal stability of the incoming fluid. These passive preheaters circulate air in a closed environment and maintain the thermal chamber through the transfer of heat to or from the fluid through the walls of the hydrodynamic piping. Keep track of temperature closely. The molding material of the temperature lowering device is applied to fill a small gap between the pipe and the preheater.

  The present invention is directed to an improved optical resonance analyzer for use specifically in lattice-coupled surface plasmon resonance (GCSPR) that allows simultaneous measurement of an array of reactive sites. In particular, the present invention enables real-time analysis of up to thousands of molecular binding interactions, as well as system fluid dynamics, temperature control, sensor scanning, and collection of data from scanned sensors. It combines many features that also provide improved control of the reaction parameters involved in the analysis.

  The analysis unit can be fully automated and has all optical scanning operations automatically controlled and implemented by software included with the unit. Basically, once the buffer, sample, and sensor are loaded into the unit, the user can enter experimental parameters such as time, temperature, fluid flow rate, etc. into a computer connected to the unit, The unit can be programmed to run the entire assay and analysis either immediately or at a set time, and can provide real-time data for binding as the assay progresses.

  In particular, the present invention is directed to a fully integrated optical grating coupled surface plasmon resonance analysis unit. In the following, particularly preferred embodiments of the invention will be described with reference to the drawings. However, the design features described can be altered or modified for a particular purpose, and the production of many alternative embodiments of the analysis unit described herein is possible in light of this disclosure That will be understood immediately. All such alterations, modifications, and additional embodiments are contemplated herein and are intended to fall within the scope of this description and the dependent claims. The following description is not intended to limit the scope of the invention in any way.

  FIG. 1 includes only the parts of the unit itself that need to be contacted by the user: the buffer / reagent bottle (50), the sample tube (40), and the sensor (or flow cell) loading door (110). 1 illustrates one embodiment of the present invention. All other functions are automatic and are controlled by a computer program using parameters and instructions entered by the user via the computer.

  Referring to FIG. 2, the unit includes a support frame (70) housed in the unit and houses a sensor unit (such as a flow cell containing sensors) via a sensor loading door (110). It includes a target area (not shown) designed to In one embodiment, the sensor unit can include a lattice-coupled surface plasmon resonance (GCSPR) chip suitable for conducting and analyzing various molecular binding interactions. A typical flow cell is a reaction zone where the sensor chip is located, one end of the reaction zone where each solution (buffer, reagent solution, sample solution, etc.) can be introduced to flow across the reaction zone. And an input port for directing fluid fluid solution from the reaction area away from the sensor chip to be collected, draining it for disposal, or for additional contact with the sensor And an output port adjacent to the end opposite the reaction zone from the input port, which can be recirculated through. The input and output ports are at the point of insertion of the flow cell into the analysis unit, both ports being in contact with the internal hydrodynamic system of the analysis unit, so that the sample container and other It is configured to establish communication with the fluid reservoir and the receptacle.

  In addition, FIG. 2 shows the relationships of many structures relevant to the present invention. In particular, FIG. 2 shows a rotating light source assembly (10), a detection assembly (20), an internal fluid dynamics peristaltic pump (62), and a greenhouse assembly containing a target area containing a sensor ( 30). Once the sensor is loaded into the unit, the light source (10) directs a beam of light over the sensor (not shown). In a preferred embodiment, the sensor is a grid coupled SPR chip having a reflective gold surface imprinted or deposited with an array of target ROIs that can be scanned by the unit's illumination and reflection detection assembly. ROIs are created from the concentration of bound halves (eg, antibodies, aptamers, single stranded DNA molecules, etc.) that can interact with analytes in a sample solution. The flow cell and fluid dynamics system introduces such a solution by flow across the surface of a sensor supporting such ROI, and receives illumination from a light source and detects the sensor image in a detector assembly Designed to be simultaneously at a stationary target position for reflection towards the detector assembly, the detector assembly provides real-time optical data indicating binding or other chemical reaction events occurring on the sensor surface. Output.

  Referring again to FIG. 2, in a particularly preferred embodiment, the light source assembly (10) is pivotally mounted to the unit frame (70). The pivoting mounting allows the light beam from the light source to move through a range of angles with respect to the target area. As an alternative, the light source can be mounted on a structure that itself is pivotally mounted to the frame. The automatic turning of the light source assembly (10) is mounted on the frame of a unit capable of accurately recording changes in the light beam angle affected by, for example, the movement of the step motor (12) on the light source assembly. Achieved by step motor (12).

  Once the sensor is in place and operation begins, the light source starts a beam of light on the sensor at a predetermined angle and continues in a circular path or pattern over a range of angles. Then point it. The light reflected from the sensor surface is directed towards a stationary detector unit (20) that includes a lens assembly (22) and a detector (24) such as a CCD camera.

  FIG. 3 is a perspective elevation view of the basic elements of the analysis unit of the present invention. In particular, FIG. 3 shows a light source assembly (10) mounted via a swivel arm (9) mounted on a shaft (15) around which a swivel arm (9) can rotate. The shaft (15) is mounted on a unit frame (not shown) so that the entire light source assembly can move in an arcuate path. Data regarding the change in position with respect to the angle of the light source is reported by some type of angular position encoder such as a rotary encoder assembly (120) that may be included at the turning point of the light source installation. The encoded angular position data is output to a computer along with the resonance data received by the detector (24), the computer applies a resonance qualification algorithm and relates to the phenomenon occurring on the sensor surface. Report. Preferably, for SPR data, the unit is used to determine the SPR curve as described in co-pending and commonly assigned US Provisional Application No. 60 / 49,2061, filed Aug. 1, 2003. The EPF algorithm is used.

  In the embodiment shown in FIG. 3, the pivotable light source arm (9) is driven by a step motor (12). The motor can be secured to the frame of the unit and includes means for meshing with the light source or pivotable light source arm. In an alternative embodiment, the stepper motor can be mounted on the light source or pivotable light source arm and can be equipped with means for engaging the stationary frame. In the embodiment shown in FIG. 3, the stepper motor is fixed to the frame of the unit, mounted at one end to the pivotable light source arm (9) via a linear slide mechanism (14), and on the opposite side Is in contact with the pivotable light source arm (9) and thus the light source assembly (10) via a linear arm (16) meshing with the stepper motor at the end of the light source. When the motor is operated, the linear arm is driven forward or backward, thereby positioning the light source at various angles with respect to the sensor (112). The linear arm (16) can be smooth or threaded, in which case the step motor includes means for receiving the threaded arm and the linear arm To operate in a rotational movement. Various other methods can also be employed including servo motors and magnetic drive systems.

  In the embodiment shown in FIG. 3, the stepper motor (12) can accurately record the change in incident light angle (Δθ) caused by its operation. Alternatively, the pivotable light source assembly can be equipped with a rotary encoder for accurate reporting of light source angle changes. Subsequently, the angle data is sent along with the reflection intensity data from the detector camera (24) to the resonance qualification algorithm as already described. In a preferred embodiment, the encoder is mounted on the swivel light source arm (9) via a swivel shaft (15) and is supported by bearings mounted in the encoder housing (120). To ensure consistent angular reading from one analysis to the next, the rotary encoder includes a built-in indexing mark to accurately determine the reference point at the start of each analysis.

  FIG. 4 shows a detailed view of one embodiment of a light source assembly (10) including a pivotable light source arm (9) of the present invention. The arm is a base plate (18) for pivotally fixing the arm to the unit, means for fixing the light source to the frame (19), and a stationary motor that operates the rotational movement of the light source assembly (10). It includes a linear slide (14) for connecting the assembly (10). Linear roller bearings (17) are shown and can be included to ensure non-vibrating movement of the assembly relative to the frame.

  In operation, the motor (12 in FIG. 3) moves the (illumination) light source in a circular path or pattern beyond the sensor (112), which continuously changes the angle of illumination that strikes the sensor (112). . Thus, changing the angle of the light source changes the angle of incident light striking the sensor. The sensor is placed at the optical apex of the angular range of the light source so that the collimated beam of light stays fixed at one position on the chip surface while the light source is moved through its angular range. Yes. This means that any spatial non-uniformity of the collimated beam intensity remains substantially fixed with respect to the area of interest (ROI) that has been immobilized on the sensor surface while the angle of incidence is scanned, And, it is particularly advantageous in the present invention that the sensor helps to ensure that it does not create distortion of the measured resonance profile that can lead to false apparent angular shifts of resonance.

  In an alternative embodiment, the light source itself can be pivotably mounted directly to the unit frame. In another embodiment, the pivot point (15 in FIG. 3) of the light source assembly can be the position of a motor or other rotational actuation means.

  As seen in FIG. 4, the light source assembly (10) includes a light source, such as an LED assembly (11), for generating illumination directed to the sensor. In one embodiment, the LED assembly includes a single 875 nm LED mounted on a printed circuit board and further includes a separate aperture element (not shown) for blocking unwanted light. However, the single wavelength light source (or narrowband wavelength light source) is not critical and is suitable for performing SPR analysis where a range of wavelengths is considered herein. An adjustment mechanism for the LED housing is preferably used to align the light source at a position away from the axis required to generate a 2 degree lateral offset angle of the collimated light source beam. Is included. Any plastic housing enclosing the LED is preferably flattened and highly polished to remove excess plastic and surface defects in front of the LED die. This allows accurate collimation and minimizes any scattering of light caused by plastic surface defects. The inventor found that the original spherical surface on the plastic enveloping many commercially available LEDs serves as a low quality lens for directing the LED beam in normal applications, but such a design is It has been discovered that degrading the collimator significantly interferes with the sharpness of the resonance profile in the SPR system. Optically flattening the plastic housing of the LED significantly and surprisingly reduced SPR noise. Thus, presenting an optically flat exterior is to procure an LED without plastic encapsulation, or to obtain an LED with a flat encapsulation, or to encapsulate the resulting LED Most preferred when using an LED light source to grind.

  As can be seen in FIG. 4, the light source also includes a light source optics assembly (13) for optimizing the characteristics of the light generated by the LED being directed to the surface of the sensor. In one embodiment, the light source optics assembly is housed in a lens tube (13) and includes a spherical aberration corrected lens for collimating light to a degree limited only by the size of the light emitting area of the LED die. In addition. The light source optics assembly typically further includes an interference filter for blocking unwanted wavelengths and a near infrared polarizer (not shown) oriented to provide P-polarized light incident on the sensor. . A conventional composite dual achromatic lens provides sufficient spherical aberration correction for this application. The lens tube also provides a means for focusing the collimating lens to optimize the collimation of the light generated by the LED.

  In a preferred embodiment of the present invention, the source beam is offset from the center line of the optical system by a short distance (eg, about 2.3 nm on the scale of the prototype device of the present invention), which is about an angle of about 2 degrees. Cause beam skew. This offset is used to reduce any ghost reflections caused by light reflecting from various optical surfaces in the instrument. In particular, non-sequential ray tracing analysis allows each point of stray light concentration in the detector plane (a “hot spot” to be discussed in more detail below) to image the optical assembly, particularly the detector assembly. It has been demonstrated that it can be the result of multiple sets of double reflections occurring between various pairs of optical surfaces in a lens. These hot spots move as the SPR angle changes over the course of the scan, resulting in additional distortion to the shape of the SPR resonance at a particular ROI, and hence inaccuracies in the SPR analysis.

  Preferred optical systems of the present invention include one or a combination of three features to minimize the occurrence of hot spots or image flare. (1) A highly efficient multilayer where all lens surfaces in the imaging lens assembly of the detector assembly (described below) are tuned to the operating wavelength of the light source, ie 875 nm in the preferred single wavelength embodiment. Coated with an anti-reflective coating, (2) as a novel aspect of the present invention, off-axis lateral beam skew, ie, the deviation of the light source described above, and (3) lateral beam A slot-shaped aperture stop, offset from the axis in the imaging lens, combined with skew. These features have been found to significantly reduce the hot spots known in prior art systems.

  As stated above, the optical analysis unit of the present invention is a precision rotation code (not shown) for accurately reporting the position of the light source and hence the angle at which the illumination is striking the surface of the sensor. Can also be included. In one embodiment, the rotary encoder is mounted on the frame of the unit and is mounted on the light source assembly at a pivotable point of the rotary encoder mounted on the frame. In a preferred embodiment, the rotary encoder includes a pivot shaft, an encoder housing, and an encoder. The rotary encoder reports data relating to the angle of the light source to a computer programmed to interpret the angular position of the transmitting light source corresponding to each camera image. This angle data and camera image data for each full-angle scan is input into an appropriate algorithm for real-time determination of the SPR resonance angle at each ROI on the sensor chip.

  As can be seen in FIG. 2, the optical analysis unit of the present invention is for measuring changes in the optical resonance angle at each of up to thousands of reaction loci or ROIs as the reaction proceeds on the surface of the array sensor. A detector assembly (20) for receiving the light reflected from the surface of the sensor and for analyzing the light.

  FIG. 5 is a cross-sectional view of a detector assembly (20) suitable for an analysis unit according to the present invention. In a preferred embodiment, the detector detects in multiple degrees of freedom, an image lens assembly (22) that includes an aperture (130) for receiving illumination reflected from the surface of the sensor (112 in FIGS. 3 and 6). Optical sensitivity such as internal (23), central (25) and external (27) gimbal seat assemblies for adjusting the instrument assembly, CCD chips (not shown) mounted in the detector A detector such as a monochromatic charge-coupled device (CCD) camera (24) including an intelligent element, a corrector plate (21) for reducing aberrations associated with the walking effect described above (eg, a CCD in a CCD camera) CCD window (28) for protection of detector sensing elements (such as chips) and for enabling operation at temperatures below the periphery of the camera by providing an inert atmosphere to the sensing elements. Beauty, including passive cold finger (26) for reducing condensation of contaminant vapors from the occurrence of nucleation locus on CCD window (28) which can be cooler than the surroundings. Such condensation points, if present, scatter light and introduce scanning angle dependent signal variations in the affected ROI.

  Preferred detectors are (a) have adequate quantum efficiency at the operating wavelength, and (b) preferred for dark signal drift and response constancy (whether or not the detector needs to be cooled) Is stable in temperature, and (c) sufficiently low background and readout noise, and sufficient analog / sound so that the signal under normal SPR measurement conditions is substantially limited to photon shot noise. Digital converter (ADC) resolution, (d) has a sufficient number of pixels to clearly resolve the sensor target ROI, and (e) can be read out fast enough to follow the angular scan rate, And (f) a CCD camera selected to have as large a pixel well electronic capacity as possible to minimize the aforementioned shot noise. More specifically, the number of pixels and the pixel well capacity should be selected to maximize the total combined charge capacity of all the pixels making up the ROI image. Although this preferred embodiment uses a CCD camera, other solid-state array cameras such as CMOS cameras can also be employed as detectors.

  In order to optimize the collection and analysis of illumination data reflected from the sensor surface, the detector assembly preferably utilizes a specially corrected large numerical aperture double telecentric lens system. “Telecentric” refers to the lens aperture stop being at infinity when viewed from the object plane or image plane. In other words, the acceptance cones from all points on the object plane (or image plane) point in the same direction, i.e. the cone axes are parallel. “Dual telecentric” means that this is true for both the object side (sensor chip) and the image side (detector sensing element). Telecentricity is not essential, but is very advantageous in the present invention, particularly on the object side. This is because all ROI undistorted images on the array must be available throughout the scan angle range. Using a telecentric design, the lens aperture can be directly mapped to the incident angle in the same shape anywhere in the object plane, and the full extent of the lens aperture stop to accommodate the angular scan range at all field points (ie , Over the entire surface of the sensor chip). As stated above, the dual telecentricity related to the present invention is also telecentric in the detector and is advantageous in the present invention. This is because the dual telecentricity minimizes the extreme incidence angle on the detector sensing element (CCD chip) and also greatly changes the detector sensitivity between different ROIs at extreme scan angles. Because it helps to avoid. In the present invention, dual telecentricity is achieved by making each half of the lens system telecentric separately, so that the intermediate aperture stop, i.e. the slot, is in object space (sensor chip) and image space (CCD). It appears to be at infinity when viewed from both sides.

  This special lens system is designed to fit the SPR sensor chip active width for the area of the selected CCD chip with a lateral magnification, and scans approximately 10 degrees (numerical aperture 0.10 on the object side). It was specifically optimized to minimize image motion (ROI shift) over the sensor range over the angular range. This optimization also takes into account the adopted narrow wavelength range as determined by the 4 nm full width at half maximum (FWHM) of the interference filter. The limited spectral bandwidth required eliminates the need for chromatic aberration control, which allows the use of a single high index glass type for all elements, thus reducing costs. The resulting residual chromatic aberration is not great. This is particularly advantageous. This is because the degree of freedom of design is not consumed and control is performed for chromatic aberration and distortion. Similarly, fewer lens elements are needed to control ROI shift aberrations because a single glass can have a refractive index greater than the average refractive index of many glasses in a color corrected system. . In fact, it is not clear that both requirements (ie, elimination of ROI shift and broadband color correction) can be achieved at any reasonable cost in an equivalent lens design. Of course, commercially available telecentric CCD lenses that are typically free of chromatic aberrations are not approaching the walk requirements achieved using the present invention.

  FIG. 6 represents a diagram of the various elements including the novel lens system within the detector assembly of the present invention. In the embodiment shown in FIG. 6, the lens assembly positioned between the target area sensor (112) and the detector sensing element (113) has an objective section (120) created with refractive elements 121-124. A slot opening (130), an imaging section (125) made of refractive elements 126-129, a corrector plate (21), and a detector window (28). The relative position of the passive cold finger elements described above with reference to FIG. 5 is indicated by element 26 in FIG. FIG. 6 shows that light strikes the sensor chip (112) from a light source (not shown), followed by the lens optics (120, 125), slot aperture (130), and corrector plate (21). It also shows the path of light (dotted line) while being reflected off the sensor through and leaving the sensor and reflected onto the detector sensing element (113) via the detector window (28).

  This special lens assembly design is particularly advantageous. Because standard “commercial” lens systems and traditional telecentric lens designs cannot maintain proper image stability on a CCD camera while the swivel arm scans through a range of angles. Maintaining this is essential for optimal performance of the optical analysis unit described herein. As outlined above, the camera identifies the region of interest (ROI) on the sensor that corresponds to the target to be analyzed. Due to higher order aberrations that depend on both the field size and aperture size in conventional lens systems, the optical image of the ROI fixed on the sensor moves across the detector surface while the illumination angle is scanned or “Walk” and increase measurement noise. This occurs in particular at off-axis field points and at large scan angle deviations from the lens axis. In conventional illuminated systems, when the full aperture stop is illuminated, rather than a small point segment as in this case, the responsible aberrations are field curvature, astigmatism, and coma. including. Importantly, the tilt of the object field (eg, typically 21 degrees in the preferred configuration here) and the tilt of the image plane resulting from the Scheimpflag principle add to the difficulty of reducing measurement noise. However, in angular scan SPR, these aberration classifications described above are not directly applicable or useful, and a numerical minimization of image shift as a function of incident angle is required in the design procedure. Note that aberrations that do not depend on both field radius and aperture angle, such as simple distortion, usually do not affect SPR results.

  The dual telecentric lens assembly described above is specially designed and optimized to solve the walking problem described herein and sandwiched between specially configured aperture stops. It includes an objective section and an imaging section, each containing four elements, each having a high-efficiency multilayer anti-reflective coating. The track length of the lens assembly in the most preferred embodiment is 272 mm.

  The double telecentric design accommodates a large angular scan range (± 5 degrees for a total mechanical displacement of 10 degrees) while minimizing the diameter of the glass elements in both the objective and imaging sections. (See FIG. 6.) Continuing to limit the diameter of the element subsequently reduces the cost and weight of the lens and facilitates aberration control.

  As shown in FIG. 6, a slot shaped aperture (130) was chosen instead of the conventional circular aperture stop. The lens has been corrected sufficiently well to allow the use of an entire circular aperture with a numerical aperture (NA) of 0.10 in object space (corresponding to 0.18 in image space). The SPR application described in does not require the use of the entire aperture. Thus, the aperture is preferably designed to reduce stray light caused by diffuse scattering from sensors and other surfaces, and to eliminate residual “hot spots” due to multiple specular reflections. . The aperture is (a) increases usability by helping to determine the exact location of the printable area when depositing ROI points on the sensor surface, and (b) an automatic point finding algorithm It also allows an increase in the spatial resolution of fiducial marks on the sensor designed to serve as a reference position for determining the position of the point itself when.

  At any one sensor illumination angle, the zero order light reflected from the sensor surface occupies a relatively small point in the aperture plane, so the instantaneous aperture is in principle a small circle centered on the current scan angle. Limited. This circle (1) encompasses a finite collimating angle of the light source and, more importantly, (2) of the small ROI section and of the reference mark and the same mark etched or otherwise sensor It needs to be large enough to avoid excessive diffractive blurring of image resolution that can interfere with faithful imaging of sensor chip ancillary structures such as identification text placed on the surface. This required circular aperture section moves linearly across the aperture plane during angular scanning so that the preferred composite minimum aperture takes the form of an elongated slot. The slot length is determined by the desired angular SPR scan length, while the slot width is selected to minimize the diffraction spurious signal in the image.

  Thus, as seen in FIG. 6, the novel aperture stop of the present invention is preferably in the form of a slot with round or square ends. This improvement minimizes stray light background and increases the apparent SPR resonance depth by eliminating diffuse scattered light at angles corresponding to unwanted portions of the lens aperture. Typical aperture widths and lengths correspond to about 3 degrees and 13 degrees, respectively, in the object space. Preferably, the slot aperture is 2 in the lateral direction to accommodate lateral beam skew employed to accommodate light source offsets and eliminate multi-reflecting hot spots as discussed above. Degree of deviation, or about 2 mm from the optical axis. In addition, the hot spots can be partially supported by a multilayer antireflective coating on the lens element. The slot opening can be either fixed in place or removable for convenience in the optical alignment procedure.

  As shown in FIG. 6, the SPR central angle, ie, the angle at which the illumination from the light source strikes the surface of the sensor chip at the center of the scan is about 21 degrees at the surface of the sensor chip. Therefore, the lens axis is set at an angle of 21 degrees from the normal to the chip surface to best accommodate the angular scan range centered at this angle. For best image quality across the field, the detector surface is tilted about 12 degrees relative to the lens axis as shown. The precise angle is selected by ray tracing optimization and is adjusted using the walk minimization criterion, but is approximately given by the well-known Scheimpflug condition.

  In accordance with the present invention, the residual ROI image shift and other aberrations resulting from field and image plane tilt to further optimize image quality across the entire field, i.e., all ROIs on the sensor chip, That is, the normal tilt of the 21 degree sensor and the normal tilt of the 12 degree detector element is additionally compensated by a flat glass corrector plate (21) of about 1 mm thickness tilted at an angle of about 20 degrees. Has been. This plate can be omitted with some degradation in image quality and walk performance, but is preferably included. In an operational embodiment of the invention, the lateral magnification is 0.57 in order to adapt the sensor chip width to the CCD chip size. Due to the slope of the field, the vertical magnification is lower, about 0.53.

  Similarly, as seen in FIG. 5 and as shown in FIG. 6, the lens assembly of the present invention is a passive cold finger for increasing the sensitivity of the detector assembly for data analysis. (26) is preferably included. In particular, airborne contaminant vapor degassed by the warmer surface in the gap between the detector window (28) and the corrector plate (21) is usually against the coldest surface, which is the detector window. Sucked. Non-uniform deposition on the detector window or corrector plate causes intensity variations that lead to increased noise and drift in the SPR signal. To solve this problem, passive cold fingers made of high conductivity metals, such as copper, are connected to a large thermal mass outside the air gap and are designed to maintain ambient temperature so that the finger is It is the coldest object in it and thereby ensures that the vapor is drawn to the fingers rather than away from the vapor to be aspirated and deposited on the detector window or corrector plate.

  As can be seen in FIG. 5, the position or orientation of the detector assembly (20) around multiple axes and along the same axis corresponds to multiple gimbal seats, i.e., internal gimbal seats fixed to the detector assembly ( 23), can be adjusted through the use of a central gimbal seat (25) fixed to the inner gimbal seat and an outer (27) gimbal seat fixed to the frame of the unit. Specifically, the gimbal has an image recording detector sensing element in a detector (24) with sensors in six directions (X, Y, Z (see FIG. 6), ρ, σ, and φ). Used to align. This alignment is necessary to precisely match the active area of the detector sensing element (eg, CCD camera chip) with the magnified and tilted image of the sensor array formed by the optics. is there. The orientation along the X or Y axis is adjusted by moving the camera with respect to the internal gimbal seat. Orientation along the Z axis (ie, the optical axis of the lens assembly) is achieved by swinging the detector position relative to the sensor position. The rotation ρ around the X axis is adjusted by the internal gimbal seat, the rotation σ around the Y axis is adjusted by the central gimbal seat, and the rotation φ around the Z axis adjusts the position of the detector with respect to the internal gimbal. By doing so, the detector assembly is adjusted, for example, by rotating it relative to the plane of the sensor. In the most preferred embodiment, the lens assembly is adjustable independently of the detector, allowing fine adjustment of the reflected sensor image reaching the detector sensing element.

  The gimbal seat is useful for adjusting the orientation of the detector assembly to optimize the fit between the sensor reflection and the image received by the detector sensing element. Once this correspondence has been optimized, the detector assembly is stationary with respect to the position of the target area containing the sensor. Thus, in a scanning operation, only the light source can move (ie, change the angle of incident light impinging on the sensor) and the sensor and detector assembly remain substantially stationary or as stationary as possible. Once adjusted, the detector assembly is locked or fixed using, for example, fixing screws, and the target area design is in the correct position for the flow cell to reflect incident illumination from the light source, and , Any fluid dynamic system incorporated in the analysis unit is fixed at a correct position to be a contact point. Sensor and detector immobilization with respect to a moving light source avoids the need to adjust the position of the active light source and the detector, and uses a compensation algorithm in post-data collection data processing to correct the image path. Avoid the need to compensate for variations.

  As seen in part in FIG. 2, the instrument of the present invention can also include a complex hydrodynamic system (60) for directing reagents and samples to the surface of the sensor. In the preferred embodiment shown in FIG. 7, after the reagent and sample contact the sensor, the hydrodynamic system allows the user to recirculate to any sensor area (112) or remove from the unit. Fluid present in the flow cell containing (112) can be directed to the waste receptacle (63) or alternatively to the collection vessel (64) or back to the sample reservoir (40) Designed to be.

  As seen in FIG. 7, reagents (eg, cleaning solutions) added to the buffer reservoir (50) integrated into the hydrodynamic system are removed from the reservoir by a peristaltic pump (62), for example. It is sucked into the system. However, any known method for drawing or propelling fluids through conduit systems such as vacuum, forced air, electrical motion, etc. can be used. In a preferred feature, the reagent is passed through a degassing system (80) integrated within the hydrodynamic system, as described above, and subsequently directed to the surface of the sensor (112). As shown in FIGS. 1 and 2, the unit can include a sample region (40) adjacent to a buffer reservoir (50). As shown in FIG. 2, the sample region (40) is integrated with the hydrodynamic system via the sample injection valve body (42). The sample region is designed to separately store several samples (eg, analytes or detection antibodies) in separate sample tubes (not shown) housed in the sample region. Like the reagent above, the sample is aspirated from one of the tubes by an injection valve body, for example via a peristaltic pump. The sample is directed to the surface of the sensor and, after contact with the surface, is further directed to a waste receptacle (63) or through the system for recirculation and re-contact with the sensor, Either returned to the original sample tube (40) for removal from the unit or to a special sample return tube (64). In addition, as shown in FIG. 7, the hydrodynamic system of the present invention is a multi-channel valve such as a six-way valve (66) as shown to direct fluid through a specific channel. Can be used. The unit can also include additional lines (65) for additional buffers by connecting with bulkhead joints (67) included in the unit.

The hydrodynamic system incorporated in the optical analysis unit of the present invention is most preferably
Also included is a “bubble rupture” degassing unit (80) capable of supplying a high speed pulsed flow driven by a syringe pump (65). Such a bubble rupture system is designed to accommodate a removable flow cell, ie, the sensor (contained in this flow cell) can be constantly changed. Used to overcome inherent problems in the system. When a new flow cell is operated, it is common for bubbles to remain in the cell gap after filling with buffer. Bubbles may be introduced accidentally during sample switching and other hydrodynamic operations. These bubbles interfere with measurements on the affected ROI and must be removed. Bubbles can be difficult to remove, particularly when the cell dimensions are reduced as in the present invention. We have observed that the application of very high fluid flow rates beyond the critical rejection flow rate (CDF) for short periods of time is due to wet tension non-uniformities at the flow cell surface or due to geometric discontinuities. It was determined that bubbles could be removed.

For example, the CDF required to remove bubbles trapped in a planar cell due to non-uniform surface characteristics on the sensor surface is such that the wet tension (τ ₂ ) downstream of the bubbles is equal to the upstream wet tension (τ to be greater than τ ₁ ), approximately given by
CDF = Wh ² {τ ₂ −τ ₁ } / (6ηL)
Where W is the cell width, h is the height of the cell gap, η is the fluid velocity, and L is the nominal length of the bubble along the flow axis. The differential wetting tension Δτ = τ ₂ −τ ₁ can be roughly estimated, and the actual values encountered in reality are usually found experimentally. Although the analysis clearly shows a geometric scale, this scale has been found to be the same for other mechanisms of bubble retention. The critical flow rate decreases rapidly as the gap height decreases, but the pressure drops as the critical flow rate increases. Similarly, if the wet tension difference persists over a small distance, the inverse dependence on bubble length indicates that very small (ie, short) bubbles can be difficult to remove.

  It has been determined that multiple applications of large flow pulses are usually necessary to eliminate bubbles while they move from one “sticky” location to the next “sticky” location. To remove any bubbles from the flow cell, the hydrodynamic system should be used to apply a “bubble rupture” or high flow rate pulse to the sensor cell via, for example, a small flow impedance fluid flow path It incorporates a positive displacement pump (syringe pump). In addition, real-time monitoring for bubbles can interrupt the hydrodynamic operation and “burst” any bubbles that may accumulate.

  SPR analysis is extremely sensitive to temperature and temperature fluctuations, so this parameter must be controlled as closely as possible. In order to maintain the stability of the system, as well as to generate accurate and consistent data regarding the binding reactions proceeding on the surface of the sensor, the user has control over the temperature of the internal environment of the disclosed unit This is very important. Thus, the user can quickly and precisely adjust the temperature in the system, adjust the temperature of reagents and samples passing through the hydrodynamic system, and control the temperature of the binding reaction on the surface of the sensor In order to make it possible, an optical greenhouse is designed for incorporation into the optical analysis unit according to the invention, as seen in FIG.

  In addition, it is desirable to allow the user to perform experiments at various temperatures both above and below the ambient temperature. To achieve this, as seen in FIG. 2, the greenhouse holds the target area where the sensor is located and contains at least part of the hydrodynamic system described above. In a preferred embodiment, the warming room includes a proportional integral derivative (PID) controlled thermoelectric module that includes a circulation fan, a cooler, a heat fuse, and a sensor.

  A preferred greenhouse is shown in FIG. The warming chamber is advantageously lined with an insulating foaming agent (32) that maintains the thermal stability within the warming chamber. This insulation protects the sensor area, incoming fluid at the sensor surface, and reactions occurring on the sensor surface against temperature fluctuations that can be caused by the environment or the operation of the instrument. Depicted in FIG. 8 is a top plate assembly (34) in a warming chamber for accurately positioning and securely holding a sensor (flow cell) in place in a target area; and It is a mechanism for transporting the sensor into the target area. The proximity of the four-way valve (36 in FIG. 7) to the sensor area is indicated by numeral 36 in the figure. An optical sensor (not shown) can be included to determine the precise positioning of the sensor (flow cell) relative to the top plate. A passive preheater (38) for maintaining the thermal stability of the incoming fluid is also shown. These passive preheaters, for example made of finned copper, are used to circulate air in a closed environment and through the walls of hydrodynamic piping embedded in grooves in the heat exchange block It closely tracks the temperature of the thermal chamber through the transfer of heat to or from the fluid by conduction. The molding material of the temperature lowering device is applied to fill a small gap between the pipe and the preheater.

  An alternative to a passive preheater is the use of active heating and cooling of the fluid stream using an additional servo loop. However, passive hydrodynamic heat exchangers are preferred because they are based on cost and simplicity and do not introduce temperature cycles or noise due to imperfect feedback control. The difficulty of using a passive heat exchanger is that a finite temperature difference is required for heat to flow to or from the heat exchanger. This difference is proportional to the required heat flux and therefore depends on the fluid flow rate, heat capacity, and the desired increase or decrease in fluid temperature.

  The ultimate achievement of the pre-heater is to bring the fluid as close as possible to the temperature of the chamber and thus to the temperature of the sensor. However, flowing heat in and out of the pipe requires a temperature difference between the fluid and the air in the greenhouse. Any difference in temperature between the fluid entering the sensor flow cell and the prescribed sensor temperature will change the desired temperature of the sensor, which poses a serious problem. The exact SPR response is highly temperature dependent. Thus, if the pre-heater had sufficient thermal mass, the pre-heater could function as a cooler or heat storage, but longer operation executions where the temperature of the fluid and the greenhouse would be extreme would be Cause final heating or cooling of the vessel, resulting in thermal SPR drift. In addition, such a large thermal mass also reduces the rapid equilibration to the required warmer temperature adjustment.

  The novel solution described herein is to separate the hydrodynamic heat exchanger for each fluid circuit into a number of sections that are thermally isolated and connected in series. According to this design, the overall size and hydrodynamic absolute volume for a segmented heat exchanger is dramatically smaller than both single stages designed to achieve the same outlet temperature error. In practice, the first such stage carries most of the total heat flow required to bring the fluid stream to the temperature of the chamber, while the temperature error is a fraction of the original value of the temperature error. Operating at a block temperature far below the temperature of the thermal chamber, and still keeps the fluid flow away from the desired temperature. The next similar stage reduces errors due to similar factors, and so on.

The performance of individual heat exchanger sections in an isothermal air stream can be evaluated as outlined below. Heat is transferred from an isothermal air stream into a finned block of highly conductive material such as aluminum or copper, and then into a fluid stream passing through a passage in the block or good thermal with the block It is conveyed through the piping in contact.
Figure: Thermal analysis of one section of air / section / water heat exchanger section

In the above scheme, Q is the thermal power being transferred from the air at temperature _TAIR to the liquid stream in the section under steady state conditions. Volumetric flow rate of the liquid F, the density [rho, and the heat capacity is c _p. The heat transfer coefficient of forced air for the finned block is h, the effective area is A, and the heat accommodation coefficient of the liquid tube is _fTA . Note that f _TA is a function of flow rate F. Solving the simultaneous equations shown in the above figure gives the following for the outlet temperature T _{OUTLET of the} section.
T _OUTLET = T _INLET + {f _TA hA / (hA + Fρc _p f _TA )} {T _AIR −T _INLET )
Where T _INLET is the inlet temperature for this particular segment.
The above equation can be rewritten for a “temperature error reduction factor” R defined below.
R≡ (T _AIR −T _OUTLET ) / (T _AIR −T _INLET )
The following is the result.
R = 1− [1 / f _TA + 1 / β] ⁻¹
Where β is a dimensionless measurement of the relative air heat transfer efficiency of the section given by:
β = hA / (Fρc _p )
In all cases, the fluid temperature termination factor f _TA (F) is constrained by:
0 <f _TA <1
And since β> 0, R is also subjected to the same following constraints.
0 <R <1

Typically, each partition is designed such that f _TA > 0.9 and β> 6, so that the value of single stage R is R <0.22. This means that an independent section of a multi-stage passive heat exchanger reduces the temperature discrepancy to about 20% or less of the previous value of the discrepancy.

For a given fluid flow rate, each heat exchanger section is always less than 1 and a fixed factor R, typically 0.20 or less, to reduce the difference between the fluid temperature and the greenhouse temperature. Function. Thus, over a number of n thermally isolated sections connected in series, the fluid temperature error is reduced geometrically as R ⁿ . Appropriate selection of the number of segments allows acceptable or even poor values for fluid temperature incompatibility to minimize or eliminate any detrimental effect on the SPR signal. It becomes possible to reduce. We have found that three to four sections are generally appropriate and provide an optimal balance between temperature equilibrium, hydrodynamic absolute volume, and flow rate.

  As stated above, in order to generate accurate SPR data using this unit, it is advantageous that the user has the ability to control the ambient temperature at which the binding reaction on the sensor surface proceeds. is there. In this regard, it is also advantageous for the user to have the ability to control the temperature of both the reagent and sample while flowing through the hydrodynamic system prior to contact with the sensor. In order to achieve this control over the temperature sensitive element of operation, it is preferred that at least a portion of any hydrodynamic system incorporated within the optical analysis unit is housed in a greenhouse, such as those described above. Preferably, the part of the hydrodynamic system housed in the greenhouse is the section closest to the place where the binding reaction takes place, i.e. close to the sensor, including the sensor itself.

  From the foregoing description, many different embodiments of SRP and other optical analysis instruments incorporating innovative features according to the present invention are possible. All such embodiments are intended to be within the scope of this invention as defined by the following claims, including obvious modifications to the specific preferred designs disclosed herein. Has been.

  All publications and documents cited above are incorporated herein by reference.

It is a figure which shows the optical resonance analysis unit (100) of this invention with which the external cover was mounted | worn. The unit design allows the user to insert the sensor (or flow cell containing the sensor chip) into the unit without removing the reagent / buffer bottle (50), sample tube (40), and outer cover. Chip door (110). The functions of all other units can be controlled by commands entered by a user into a computer connected to the unit. FIG. 2 shows the unit of FIG. 1 with the cover removed. The main components of the unit are visible, including the swivel arm, the detection assembly (20), and the light source assembly (10) including the warming chamber (30). FIG. 2 shows one embodiment of a light source assembly (10) attached to a swivel light source arm (9) and associated with a detector assembly (20). The arrow is reflected in the direction of illumination from the light source (11) impinging on the sensor (112) (down arrow) and the surface of the sensor (112) and in the direction of the detector assembly (20) (up arrow) Indicates. FIG. 3 is a view of a swivel light source arm (9) with a light source (11, eg, an LED assembly) and a light source optical system assembly (13) mounted on the light source. A linear slide (14) for connecting the swivel arm (9) to a driver motor (12) (not shown), a base plate (18) of the swivel arm (9), a roller bearing (17), and A light source assembly support (19) for securing the light source (11) to the swivel arm (9) is also shown. Lens assembly outer case (22), inner, middle and outer gimbal seats (23, 25 and 27, respectively), corrector plate (21), passive cold finger (not shown) 26) is a cross-sectional view of the detector assembly (20) showing both ends of 26), a detector window (28, eg, a CCD window), a detector (24, eg, a CCD camera), and a slot opening (130). . FIG. 6 is a diagram of the various elements of the detector optics located within the lens assembly (accommodated in the outer case 22 shown in FIG. 5) of the detector. The elements of the lens assembly are optically sensitive elements of the objective section (125), slot aperture (130), imaging section (125), corrector plate (21), and detector (113, eg, a CCD chip). Immediately adjacent detector window (28) is included. Accordingly, the lens assembly is sandwiched between the sensor (112) and a light receiver for the sensor reflection image, ie the sensing element (113) of the detector. The relative position in the alignment of the elements in the lens assembly of the passive cold finger (26 in FIG. 5) is indicated by the numeral 26. X, Y, and Z represent the plane of motion of the detector assembly controlled by the gimbal seat. The location of the buffer container (50) and the sample solution container (40), as well as directing each solution through the unit to the target area (including the sensor 112) and into the original sample tube (40) FIG. 6 is a hydrodynamic view showing the various paths that can be directed back to the collection tube (64) or to the waste receptacle (63). The sensor is typically housed in a flow cell, and the flow cell constructed using input and output ports that are in contact with the unit's hydrodynamic system is routed through an entry door (110 in FIG. 1). A changeable device inserted in the optical analysis unit (100). FIG. 7 also shows the relative position of the warmer chamber (30) including the target area including the preheater (38), the four-way valve (36), and the sensor (112). The location of the warmer enclosure and the location of the preheater (38), the (hidden) four-way valve (36), the top plate assembly (34), the thermal insulation panel (32), and the sensor chip (112) FIG. 2 is a partially cutaway perspective view of a portion of an optical analysis unit according to the present invention showing a carrier for

Claims (22)

An optical analytical instrument in the form of an array surface plasmon resonance (SPR) analytical instrument,
(A) a reflective SPR sensor array;
(B) A light source set configured to irradiate the reflective SPR sensor array with a parallel light beam to give a reflective array image of the reflective sensor array and to scan the incident angle of the parallel light beam within a certain angle range. Solid,
(C) a detector assembly oriented to receive a reflective array image of the reflective SPR sensor in the angular range, wherein the two-dimensional detector sensing element is tilted with respect to the optical axis of the lens in accordance with a Scheimpflug condition And a detector assembly including a lens assembly for focusing a reflective array image of the reflective SPR sensor onto the tilted detector sensing element;
Objective section configured to produce a real image of the tilted reflective array at infinity, followed by an image for converting the real image of the tilted reflective array into a tilted real image on the tilted detector sensing element. A dual telecentric lens assembly for sensor imaging including a forming section, wherein the objective section and the imaging section share a common intermediate aperture plane located in the air gap therebetween, and the angle SPR An optical analysis instrument in which a slot-type aperture stop having a length determined by a scanning length is sandwiched .   The optical analysis instrument of claim 1, wherein the lens assembly includes a corrector plate.   The optical analysis instrument of claim 1, wherein the lens assembly includes a passive cold finger.   The optical analysis instrument of claim 1, wherein the sensing element is a CCD chip.   The optical analysis instrument of claim 1, wherein the reflective SPR sensor is a grating coupled surface plasmon resonance (SPR) sensor.   The optical analyzer according to claim 1, wherein the light source assembly includes a light source and a light source optical system for collimating light emitted from the light source. The optical analysis instrument of claim 6 , wherein the light source is one or more light emitting diodes (LEDs). The optical analysis instrument according to claim 7 , wherein the light source is an LED that emits a narrow band of wavelengths. The one or more LEDs are encapsulated in a plastic material, and the plastic material is optically flat or optically flat to minimize optical distortion caused by the plastic material. The optical analysis instrument according to claim 7 , wherein   The optical analysis instrument according to claim 1, wherein the light source assembly is provided on a pivot arm that is pivotable to scan an incident angle of the parallel light beam in a certain angle range. The optical analysis instrument according to claim 10 , wherein the pivot axis of the pivot arm is substantially at the center of the reflective SPR sensor array. The optical analysis instrument according to claim 6 , wherein the light beam emitted from the light source is shifted from a central axis of the light source optical system. 13. The optical analysis instrument according to claim 12 , wherein the light beam is offset from a central axis of the light source optical system so as to give a transverse beam skew of 2 degrees. The optical analysis instrument according to claim 6 , wherein the light source optical system is fixed, and the light source is movable so as to scan an incident angle of a parallel light beam in a certain angle range. The light source optical system and the light source is fixed, the light source is configured to illuminate the pivotable mirror for scanning at an angle range of the incident angle of the parallel light beam, to claim 6 The optical analysis instrument described. Wherein optionally the light source optical system is fixed, the light source optical system is comprised of a linear array of light source optical system that can be sequentially illuminated for scanning at an angle range of the incident angle of the parallel light beam, according to claim 6 Optical analysis equipment. (G) a hydrodynamic system,
(I) one or more solution storage tanks and / or solution input connections;
(Ii) supply tube piping connecting the one or more storage tanks and / or input connections to the target area;
(Iii) a waste receptacle, a solution reservoir, a collection container, and a removal tube line connecting the target area to one or more elements selected from the group of target areas;
The optical analysis instrument of claim 1, further comprising a fluid dynamics system comprising (iv) one or more pumps for propelling fluid through the supply and removal tubing. The optical analysis instrument according to claim 17 , wherein the hydrodynamic system (g) further includes bubble bursting means for water-washing trapped air bubbles from the hydrodynamic system. The optical analysis instrument according to claim 17 , wherein a part of the hydrodynamic system and the target area are accommodated in a greenhouse. 20. The optical analysis instrument of claim 19 , wherein the temperature of the fluid transferred to the target area is controlled using one or more passive heat exchangers. 21. The optical analysis instrument of claim 20 , wherein the cooling device comprises a series of segmented passive heat exchangers. 20. The optical analysis instrument of claim 19 , wherein the temperature of the fluid transferred to the target area is controlled using one or more active heating or cooling loops.

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